Quantitative Determination of Principal Aporphine and Benzylisoquinoline Alkaloids Due to Blooming State in Lotus Flower (Flower Buds of Nelumbo nucifera ) and Their Hyaluronidase Inhibitory Activity

Abstract

Using a recently developed analytical protocol, distributions of 5 aporphine alkaloids, nuciferine (1), nornuciferine (2), N-methylasimilobine (3), asimilobine (4), and pronuciferine (5), and 5 benzylisoquinoline alkaloids, armepavine (6), norarmepavine (7), N-methylcoclaurine (8), coclaurine (9), and norjuziphine (10), in lotus flowers (the flower buds of Nelumbo nucifera) were analyzed. The flowers were collected at different blooming states (beginning of blooming, one-third in bloom, half in bloom, three-quarters in bloom, and in full bloom) from Saga prefecture, Japan (NN-S1–5). The samples from the beginning of blooming state (NN-S1, 16.35 mg/g in dried material) were found to possess the richest total alkaloid content (1-10). The samples of half in bloom (NN-S3, 52.69 mg per flower of dried material) had the highest total alkaloid content per flower. Among the alkaloid constituents, nornuciferine (2, IC₅₀ = 22.5 µM), asimilobine (4, 11.7 μM), norarmepavine (7, 26.4 μM), coclaurine (9, 11.4 μM), and norjuziphine (10, 24.3 μM) exhibited hyaluronidase inhibitory activity, which was more potent than that of the antiallergic medicine disodium cromoglycate (DSCG, 64.8 μM).

Lotus flower (the flower buds of Nelumbo nucifera Gaertn., Nymphaeaceae) is extensively cultivated in east Asian countries and has been used for the treatment of vomiting blood, bleeding caused by internal and external injuries, and various skin diseases, along with use as a sedative and anti-inflammatory agent in traditional Asian medicines. ^1,2 In the course of our exploratory studies of bioactive constituents in the flower buds of N. nucifera, we have reported that a practical liquid chromatography-mass spectrometry (LC-MS)-based method was developed for the simultaneous quantitative determination of 5 aporphine alkaloids, nuciferine (1), nornuciferine (2), N-methylasimilobine (3), asimilobine (4), and pronuciferine (5), and 5 benzylisoquinoline alkaloids, armepavine (6), norarmepavine (7), N-methylcoclaurine (8), coclaurine (9), and norjuziphine (10), in the lotus flower (Figure 1). Among these alkaloids, aporphine alkaloids, viz. nuciferine (1, IC₅₀ = 7.1 µM), nornuciferine (2, 3.9 µM), armepavine (6, 6.5 µM), norarmepavine (7, 7.5 µM), N-methylcoclaurine (8, 6.5 µM), and coclaurine (9, 3.9 µM), were found to inhibit theophylline-stimulated melanogenesis activity in murine B16 melanoma 4A5 cells, without notable cytotoxic effects at the effective concentrations. Furthermore, excellent correlations between the total 10 alkaloids (value reduced to 1) and the melanogenesis inhibitory activities (1/IC₅₀) of the lotus flower extracts have been observed. ¹ To continue the study for efficient quality control measurements, in order to ensure the authenticity and content of the active constituents as possible cosmetic ingredients, the comparative quantitative analysis for the different blooming states (eg beginning of blooming, one-third in bloom, half in bloom, three-quarters in bloom, and in full bloom) was discussed. We also evaluated hyaluronidase inhibitory activity, which seems to be effective for suppressing allergies and inflammation, as a new bio-function for the cosmetics of lotus flower extract and its principal alkaloid constituents (1-10).

Figure 1

Aporphine and benzylisoquinoline alkaloids (1-10) from flower buds of N. nucifera.

In total, 5 test samples were collected according to the different blooming states: the beginning of blooming (abbreviated as NN-S1), one-third in bloom (NN-S2), half in bloom (NN-S3), three-quarters in bloom (NN-S4), and in full bloom (NN-S5) from the same field in Saga prefecture, Japan (Figure 2).

Figure 2

Collected samples sorted according to the different blooming states at the same field in Saga prefecture (Minato, Karatsu), Japan.

As shown in Figure 3, a typical liquid chromatography-mass spectrometry (LC-MS) chromatogram under optimized conditions for alkaloids (1-10) in a standard mixture by UV (260 nm) and MS detection using ESI MS under the positive ion mode. It was found that each peak was NN-S1: beginning of blooming; NN-S2: one-third in bloom; NN-S3: half in bloom;NN-S4: three-quarters in bloom; NN-S5: in full bloom observed at the following retention times (t _R) and quasimolecular ion peaks ([M + H]⁺): nuciferine (1, t _R: 43.1 minutes, m/z 296), nornuciferine (2, t _R: 39.5 minutes, m/z 282), N-methylasimilobine (3, t _R: 29.7 minutes, m/z 282), asimilobine (4, t _R: 21.3 minutes, m/z 268), pronuciferine (5, t _R: 13.9 minutes, m/z 312), armepavine (6, t _R: 16.9 minutes, m/z 314), norarmepavine (7, t _R: 15.9 minutes, m/z 300), N-methylcoclaurine (8, t _R: 9.9 minutes, m/z 300), coclaurine (9, t _R: 8.3 minutes, m/z 286), and norjuziphine (10, t _R: 18.8 minutes, m/z 286). These peaks were unambiguously assigned by comparing their retention times with those of authentic specimens. The analytical parameters such as linearity, limits of detection and quantitation, and accuracy, etc have been examined in our previous report. ¹ According to the established protocol, contents of these alkaloids (1-5) in NN-S1-S5 were examined (Figure 3). As shown in Table 1, a distinct trend was observed with contents as follows: (1) N-methylcoclaurine (8, 0.92-5.49 mg/g in dried material) had the richest content among the 10 alkaloids (1-10) through all of the blooming states; (2) the sample NN-S1 (beginning of blooming, 14.94 mg/g) had the most abundant total alkaloid content; (3) as the blooming states, progressed, the total alkaloid content decreased; (4) comparison of the content ratio of nuciferine (1) and nornuciferine (2) in the flower buds originating in Japan (NN-S1-S5, circa 3:1-5:1) with those in Thailand (NN-1, circa 1:1) and Taiwan (NN-5, circa 2:1) showed that nuciferine (1) content was observed at a higher ratio in Japan. To characterize the efficient preparation of the extract with high total alkaloid content, the total alkaloid content per flower was considered. The half in bloom (NN-S3, 52.69 mg per flower in dried material) had the highest total alkaloid content.

Figure 3

LC-MS chromatograms of alkaloids (1-10). Selected ion monitoring (SIM) chromatograms (positive electrospray ionization [ESI]) of (a) a standard solution mixture (each 10 mg/mL) and the methanol extract of (b) NN-S1 (beginning of blooming), (c) NN-S2 (one-third in bloom), (d) NN-S3 (half in bloom), (e) NN-S4 (three-quarters in bloom), and (f) NN-S5 (in full bloom).

Table 1

Contents of Alkaloids (1-10) in the Methanol Extracts From Lotus Flowers.

Sample no.	Average of flower (g) a	Loss of drying (%) b	Extraction yield (%) c	Alkaloid contents (mg/g in dry material)
				1	2	3	4	5	6	7	8	9	10	Total
NN-S1	2.11	8.62	16.9	3.33(7.02)	1.14 (2.41)	0.15 (0.32)	0.25 (0.53)	1.04 (2.20)	0.73 (1.55)	1.68 (3.55)	5.49 (11.58)	0.39 (0.82)	0.74 (1.56)	14.94 (31.52)
NN-S2	3.22	7.99	18.4	2.52 (8.11)	0.74 (2.39)	0.10 (0.31)	0.14 (0.44)	0.29 (0.95)	0.63 (2.02)	1.13 (3.62)	5.26 (16.95)	0.43 (1.38)	0.51 (1.65)	11.75 (37.83)
NN-S3	6.12	7.97	20.3	2.00 (12.22)	0.57 (3.48)	0.08 (0.51)	0.14 (0.87)	0.12 (0.75)	0.43 (2.64)	0.81 (4.93)	3.75 (22.93)	0.35 (2.17)	0.36 (2.22)	8.61 (52.69)
NN-S4	7.85	8.97	22.0	0.93 (8.15)	0.19 (1.70)	0.06 (0.48)	0.06 (0.55)	0.01 (0.10)	0.26 (2.31)	0.48 (4.19)	1.83 (15.97)	0.23 (2.04)	0.18 (1.53)	4.23 (33.21)
NN-S5	10.42	8.67	23.7	0.48 (5.05)	0.09 (0.92)	0.03 (0.34)	0.02 (0.25)	n.d. f (n.d. f )	0.15 (1.51)	0.21 (2.24)	0.92 (9.63)	0.06 (0.95)	0.08 (0.81)	2.08 (21.67)
NN-1 d 1	—	10.33	15.0	1.76	1.75	0.07	0.63	0.69	0.83	1.45	5.73	1.30	0.75	14.96
NN-5 e 1	—	8.08	16.3	0.56	0.27	n.d. f	0.04	n.d. f	0.23	0.34	1.74	0.19	0.16	3.53

Total alkaloid content per one flower (mg/g in dry material) is given in parentheses.

^aEach fresh sample was weighed after drying by hot air dryer at 50°C for 3 days (n = 4–8).

^bEach powdered sample was dried at 105°C for 8 hours.

^cEach powdered sample was extracted twice with methanol under reflux for 120 minutes.

Originating in ^dThailand or ^eTaiwan. ¹

^fLess than the quantitation limit.

Hyaluronidase is a mucopolysaccharide related to inflammation by the histamine released from mast cells. Hyaluronidase inhibitors seem to be effective in suppressing allergies and inflammation. ^{3 -5} It is known that the antiallergic medicine DSCG exhibits a strong inhibitory effect against hyaluronidase. ³ Therefore, it has been found that there is a close relationship between allergic reactions and hyaluronidase inhibitory activity ^3,5 Through our continued studies on antiallergic principles with hyaluronidase inhibitory activity, the methanol extracts of the lotus flower, such as NN-S1 and NN-S2 (IC₅₀ = 52.8 and 58.6 µg/mL), were found to show the activity displayed in Supplemental Table S1. To identify the active constituents of this plant material, the inhibitory effects of the alkaloids (1-10) against hyaluronidase were examined. As a result, nornuciferine (2, IC₅₀ = 22.5 µM), asimilobine (4, 11.7 µM), norarmepavine (7, 26.4 µM), coclaurine (9, 11.4 µM), and norjuziphine (10, 24.3 µM) were found to exhibit hyaluronidase inhibitory activity, which was more potent than that of the anti-allergic medicine DSCG, 64.8 µM. As for the structural requirements of aporphine and benzylisoquinoline alkaloid for the hyaluronidase inhibitory activity, the results were as follows: (1) N-methyl moiety reduced the inhibitory activity [nornuciferine (2, IC₅₀ = 22.5 µM) >nuciferine (1, > 100 µM), asimilobine (4, 11.7 µM) >N-methylasimilobine (3), norarmepavine (7, 26.4 µM) >armepavine (6, > 100 µM), coclaurine (9, 11.4 µM) >N-methylcoclaurine (8, > 100 µM)] and (2) as the phenol groups increased in number, the inhibitory activities became stronger [asimilobine (4, IC₅₀ = 11.7 µM) >nornuciferine (2, 22.5 µM), coclaurine (9, 11.4 µM) >norarmepavine (7, 26.4 µM)].

In conclusion, using the recently developed analytical protocol, distributions of 5 aporphine alkaloids, nuciferine (1), nornuciferine (2), N-methylasimilobine (3), asimilobine (4), and pronuciferine (5), and 5 benzylisoquinoline alkaloids, armepavine (6), norarmepavine (7), N-methylcoclaurine (8), coclaurine (9), and norjuziphine (10), in lotus flowers (the flower buds of N. nucifera), collected at different blooming states [beginning of blooming, one-third in bloom, half in bloom, three-quarters in bloom, and in full bloom in Saga prefecture, Japan (NN-S1-5)], were analyzed. Among them, the sample from the beginning of blooming (NN-S1, 16.35 mg/g in dried material) was found to possess the richest total alkaloid content (1-10). As for the content per flower, the sample from the half in bloom (NN-S3, 52.69 mg per flower in dried material) had the highest. Therefore, when the alkaloid content was taken into consideration, it was suggested that the period of the half in bloom state is the most efficiently obtained. In addition, several alkaloid constituents, nornuciferine (2), asimilobine (4), norarmepavine (7), coclaurine (9), and norjuziphine (10) were found to exhibit hyaluronidase inhibitory activity (IC₅₀ = 11.4–26.4 µM), which was more potent than that of the anti-allergic medicine (DSCG, 64.8 µM). Several structural requirements of aporphine and benzylisoquinoline alkaloids were suggested for the hyaluronidase inhibitory activity.

Experimental

Plant Material

Five samples were collected in Minato, Karatsu, Saga prefecture, Japan from 8:30 to 9:30 am on August 10, 2016, and were abbreviated as follows: NN-S1 (beginning of blooming), one-third in bloom (NN-S2), half in bloom (NN-S3), three-quarters in bloom (NN-S4), and in full bloom (NN-S5). These plant materials were supplied by Mr Youichi Matsuura (Iwase Cosfa Co., Ltd., Osaka, Japan) and identified by one of the authors (M.Y.). Voucher specimens of them are on file in our laboratory. The materials were finely air-dried at 50°C for 3 days using a hot-air dryer.

Reagents and Chemicals

Acetic acid, acetonitrile, and distilled water were purchased from Nacalai Tesque Inc. (Kyoto, Japan). The reference sample of the hydrochloride of each alkaloid (1-10) was prepared according to the procedure described previously. ¹ All other chemicals were purchased from either FUJIFILM Wako Pure Chemical Industries, Ltd., (Osaka, Japan) or Nacalai Tesque Inc.

LCMS Analysis for Alkaloids (1-10)

An LC-20A series Prominence HPLC system (Shimadzu Co.) was equipped with a binary pump, a degasser, an autosampler, a thermostated column compartment, a UV detector, and a control module connected with a LCMS-2010EV mass spectrometer (Shimadzu Co.) equipped with an ESI interface. The chromatographic separation was performed on a Cosmosil πNAP column (5 µm particle size, 2.0 mm i.d. × 150 mm, Nakalai Tesque Inc.) operated at 40°C with mobile phase A (acetonitrile) and B (H₂O containing 0.2% acetic acid). The gradient program was as follows: 0 minutes (A:B 15:85, v/v) → 20 minutes (18:82, v/v) → 50 minutes (50:50, v/v). The flow rate was 0.2 mL/min and the injection volume was 2.0 µL. The detections were performed at 260 nm (UV) and under SIM by a positive-mode ESI-MS. The operating parameters for MS detection were as follows; nebulizing gas flow: 1.5 L/min, drying gas pressure: 0.15 MPa, CDL temperature: 250°C, block heater temperature: 250°C, interface voltage: −3.5 kV, CDL voltage: constant-mode, Q-array DS and RF voltage: Scan-mode. ¹

Preparation of Standard Solution

A standard solution was prepared according to the previously reported method. ¹ Thus, an accurately weighed 2.00 mg of each hydrochloride salt of alkaloid (1-10) was introduced into a 20 mL volumetric flask, and the volume was made up with methanol; the solution being used as a stock standard solution (100 µg/mL). Aliquots of 50, 100, 500, 1000, and 5000 µL of the stock standard solution were transferred into 10 mL volumetric flasks and the volume was made up with methanol for use as working solutions (0.5, 1.0, 5.0, 10, and 50 µg/mL, respectively) for constructing calibration curves. For calibration, an aliquot of 2.0 µL of each solution was injected into the LC-MS system. Each peak was observed at the following retention times: 1 (t _R: 43.1 minutes), 2 (t _R: 39.5 minutes), 3 (t _R: 29.7 minutes), 4 (t _R: 21.3 minutes) and 5 (t _R: 13.9 minutes), 6 (t _R: 16.9 minutes), 7 (t _R: 15.9 minutes), 8 (t _R: 9.9 minutes), 9 (t _R: 8.3 minutes), and 10 (t _R: 18.8 minutes).

Sample Preparation

Pulverized sample powders were accurately weighed (circa 2.0 g, conversion with loss on drying) and extracted twice with 20 mL of methanol under reflux for 120 minutes. After centrifugation of the combined extracts at 3000 rpm for 5 minutes, the supernatants were combined and diluted to 100 mL with the extraction solvent. An aliquot (1 mL) of the extract solution was transferred into a 5 mL volumetric flask and made up to the volume with methanol. The solution was filtered through a syringe filter (0.45 µm), and an aliquot of 5.0 µL was subjected to the LC-MS analysis. The remaining extraction solution (90 mL) was evaporated in vacuo to calculate the extraction yields.

Hyaluronidase Inhibitory Activity

The experiment was performed according to a previously reported method ^{3 -7} with a slight modification. Briefly, the assay was performed in 96-well microplates. Pre-incubation of 10 µL hyaluronidase enzyme (Type IV-S from bovine testes; 340 NF unit/mL, Sigma-Aldrich Co. LLC, St Louis, USA) or a blank buffer (0.1 M acetate buffer, pH 3.5) with 20 µL of sample or control was incubated at 37°C for 20 minutes. Twenty μL of calcium dichloride (final concentration: 2.0 mM) was added to the buffer, and the mixture was incubated at 37°C for 40 minutes. Then, 50 µL of hyaluronic acid potassium salt (final concentration: 0.6 mg/mL, Sigma-Aldrich Co. LLC) was added, and the mixture was incubated at 37°C for 40 minutes. The reaction was stopped by adding 0.4 M NaOH (10 µL) and 0.08 M borate solution (pH 9.1, 10 µL) and immediately heated by boiling water for 3 minutes. Twenty μL of the reaction solution was transferred to another 96-well microplate. Eighty μL of p-dimethylaminobenzaldehyde (final concentration: 8.0 mg/mL, Wako Pure Chemical Industries Ltd., Osaka, Japan) acetate solution was added to the reaction mixture and incubated at 37°C for 20 minutes. The optical density (O.D.) of the reaction mixture was measured using a microplate reader (SH-9000, CORONA) at a wavelength of 585 nm (reference 670 nm). The final concentration of dimethyl sulfoxide (DMSO) in the test solution was 1.0% and no influence of DMSO on the inhibitory activity was detected. All the experiments were performed in quadruplicate and IC₅₀ was determined graphically. DSCG, a clinically used antiallergic medicine, was used as the reference compound.

$$\begin{gathered} {\displaystyle \mathrm{I}\mathrm{n}\mathrm{h}\mathrm{i}\mathrm{b}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}(\mathrm{\%})=[(\mathrm{O}.\mathrm{D}.(\mathrm{C})-\mathrm{O}.\mathrm{D}.(\mathrm{N}))-(\mathrm{O}.\mathrm{D}.(\mathrm{T})-\mathrm{O}.\mathrm{D}.(\mathrm{B}))] \\ /(\mathrm{O}.\mathrm{D}.(\mathrm{C})-\mathrm{O}.\mathrm{D}.\mathrm{N}))\times 100} \end{gathered}$$

Control (C): enzyme (+), test sample (–); Test (T): enzyme (+), test sample (+); Blank (B): enzyme (–), test sample (+); Normal (N): enzyme (–), test sample (–)

Supplemental Material

Supplemental Table S1 - Supplemental material for Quantitative Determination of Principal Aporphine and Benzylisoquinoline Alkaloids Due to Blooming State in Lotus Flower (Flower Buds of Nelumbo nucifera) and Their Hyaluronidase Inhibitory Activity

Supplemental material, Supplemental Table S1, for Quantitative Determination of Principal Aporphine and Benzylisoquinoline Alkaloids Due to Blooming State in Lotus Flower (Flower Buds of Nelumbo nucifera) and Their Hyaluronidase Inhibitory Activity by Toshio Morikawa, Shuhei Okugawa, Yoshiaki Manse, Osamu Muraoka, Masayuki Yoshikawa, and Kiyofumi Ninomiya in Natural Product Communications